WO2008152144A1

WO2008152144A1 - Method and probe/primer system for the 'real time' detection of a nucleic acid target

Info

Publication number: WO2008152144A1
Application number: PCT/EP2008/057512
Authority: WO
Inventors: Werner Lehmann; Henning Hanschmann; Mandy Syring
Original assignee: Attomol Gmbh Molekulare Diagnostika; Fachhochschule Lausitz
Priority date: 2007-06-13
Filing date: 2008-06-13
Publication date: 2008-12-18
Also published as: ES2538100T3; US20100203572A1; EP2167685A1; EP2167964A1; EP2167964B1; EP2167685B1; WO2008152145A1

Abstract

The invention relates to a method for the 'real time' detection of a nucleic acid target. The invention also relates to a primer/probe system and to a kit for detecting a nucleic acid target and the use of said system and kit in a solution or array, preferably for liquid phase PCR and multiplex PCR. The method comprises the steps: use of at least one primer, which comprises at least one sequence (target bonding sequence) that is specific to the nucleic acid to be amplified and that has modifications at the 3' end, allowing an enzymatic extension that complements the target; introduction of a fluorophore 1; use of at least one probe having a fluorophore 2 at the 3' end, the probe or probes being able to hybridise with the strand of the primer that has been extended to form the target sequence at the 3' end. One of the two fluorophores is excited by light of a suitable wavelength, causing a FRET to take place between fluorophores 1 and 2, said transfer being detected by optical fluorescence imaging.

Description

Method and probe / primer system for "real time" detection of a nucleic acid target

The invention generally relates to the field of nucleic acid amplification. In particular, it relates to a method for "real time" detection of a nucleic acid target The invention also relates to a primer / probe system and a kit for detecting a nucleic acid target and its use in a PCR.

Methods for detecting and quantifying nucleic acids are known. Starting from an mRNA to be analyzed, first a single-stranded cDNA is produced by means of a reverse transcriptase, with the aid of a PCR then a double-stranded DNA amplification product is produced. The PCR can be used for both qualitative and quantitative statements. Quantification of PCR products can be done in different ways. Newer detection methods make it possible to measure the in-vitro synthesis of PCR products "real time", ie homogeneously directly in the PCR reaction vessel used in each case.This so-called "real time" PCR is a particularly sensitive method In each cycle of the PCR, the development of PCR products is monitored. The measurement of the amplification is usually carried out in thermocyclers, which have additional means for measuring fluorescence signals during the amplification reaction. The amplification products are detected, for example, by fluorescence-labeled hybridization probes which emit fluorescence signals only when bound to the target nucleic acid or, in certain cases, also by double-stranded DNA-binding fluorescent dyes. A defined signal threshold is determined for all the reactions analyzed and the cycle number Cp necessary to reach the threshold is determined for both the target nucleic acid and the reference nucleic acids. Based on the Cp values obtained for the target nucleic acid as well as the reference nucleic acid, either absolute or relative copy numbers of the target molecule can be determined.

A recent method for detecting a specific nucleic acid molecule uses so-called "Molecular Beacons" (Tyagi et al., US 6,150,097A) Molecular Beacons are dye-labeled oligonucleotides having a stem-loop structure At the two free ends of the stem segments (FIG. the 3'- and the 5'-end) is in each case coupled to a fluorophore, wherein the one as reporter dye and the other as a quencher dye which quenches the fluorescence of the reporter dye with sufficient spatial proximity by Foerster energy transfer Sequences of the stem portions at both ends of the molecular beacons are chosen so that, when the molecular beacon folds, the stem portions hybridize exclusively to each other but not to other portions of the oligonucleotide. The distance between reporter and quencher dye is sufficiently small in the state of the hybridized stem portions, so that the fluorescent dye does not fluoresce even with appropriate excitation with light. The loop portion has a sequence that is complementary to the sequence of a target sequence. When the molecular beacons and target sequence-containing nucleic acid molecules are in solution, the loop portions and the target sequence portions may hybridize, thereby unfolding the molecular beacon resolving the hybridization of the two stem portions. This unfolding leads to an increase in the spatial distance between the reporter dye and the quencher dye and the reporter dye is excited to fluoresce. With continuous observation of fluorescence intensity, an increase in intensity can be detected as the molecular beacons detect and hybridize to the target sequences. Thus, the nucleic acid molecules can be detected quantitatively. These molecular beacons have the major drawback of a costly and expensive synthesis, since these probes must be labeled with both the fluorescence and the quencher dye. Quencher fluorescent dye systems are used in comparable probe systems such as Amplifluor primers, Scorpions and TaqMan probes.

Another well-known system is the Light Cycler system. Here, the fluorescent dyes are distributed to two different oligonucleotides. At the 3 'end of the first oligonucleotide is the one fluorescent dye, e.g. Fluorescein, and the downstream annealing oligonucleotide has at the 5 'end another fluorescent dye, e.g. LC Red 640. Fluorescein is excited by an LED as a light source and emits light, which excites the second fluorescent molecule via fluorescence resonance energy transfer (FRET). The light emitted by the second dye is detected by a corresponding filter. An excitation of the second dye can only take place if the two dye molecules are in spatial proximity (distance 1 to 5 nucleotides) due to the attachment of the two oligonucleotides to their complementary strand. The principle of this system is based on the secondary excitation of a second fluorescent dye, which is only made possible by the generation of the PCR product.

The systems mentioned can basically be used for multiplex applications, but have the disadvantage that - should different target sequences are detected side by side - the reaction approach is relatively expensive because of the increasing number of different detection probes and the increasing number of probes may affect the amplification reaction. In addition, not enough FRET pairs are known to achieve a high degree of multiplexing. The previously existing probe systems also make it possible not to analyze larger sequence sections of the target sequence, without having to synthesize several fluorescently labeled, and thus costly, probes. The detection of multiple target sequences also involves the simultaneous detection of a target sequence and an internal standard or multiple internal standards in a PCR, wherein the multiple standards for the calibration of the quantification of the target sequence may also be present in different concentrations.

It was therefore an object of the invention to provide methods and primer / probe systems which can be synthesized more cheaply and simply and which have the same or improved sensitivity. By suitable modification of the primer structures, a possibility should be found of circumventing the expensive use of specific probe oligonucleotides, whereby the multiplex application, which is becoming increasingly important for today's diagnostics, should not be impaired, even if larger sequence regions are analyzed.

According to the invention, the object is achieved by a method for "real-time" detection of nucleic acid targets as products of a nucleic acid amplification reaction by means of a new primer / probe system.This method comprises the following steps: a) Use of at least one primer which comprises at least one for the nucleic acid-specific sequence to be amplified (TSS) and which permit enzymatic extension complementary to the target at the 3 'end b) introduction of a fluorophore 1. In one embodiment, this fluorophore 1 may be present directly on a first or further primer Alternatively, it may also be present on a probe c) After enzymatic extension, at least one probe is used to detect the target, which carries a fluorophore 2 at least at the 3 'end at least one probe with the strand of the target sequence at the 3 ' End extended primer carrying the fluorophore 1 hybridize or directly to target non-specific portions of the probes.

In this case, fluorophores 1 and 2 reach a sufficient proximity for the FRET, so that upon excitation by light of corresponding wavelength, a detection of the fluorescence allow the acceptor. That is, one of the two fluorophores is excited by light of a suitable wavelength, so that a FRET takes place between fluorophore 1 and 2 which is detected by fluorescence optics. Such a sufficient proximity between the two fluorophores is present, for example, when the two fluorophores 1 and 2 are not more than 10 nucleotides apart, preferably not more than 5 nucleotides, most preferably the distance between fluorophore 1 and 2 is one to five nucleotides. It does not matter whether fluorophore 2 comes to lie downstream or upstream of fluorophore 1 at the appropriate distance. Surprisingly, the FRET between fluorophore 1 and 2 is also not affected by the fact that according to the invention fluorophore 1 and fluorophore 2 are positioned on opposite strands.

Means and ways for excitation of the fluorophores with light of suitable wavelength and devices and methods for the detection of the fluorescence after FRET are known to those skilled in the art.

Likewise, those skilled in the art techniques, means and ways known primers and probes according to the invention to produce and optionally provided with Fluorophori or 2. Labeling reactions for covalent coupling of primers or probes to corresponding fluorophores are well known to those skilled in the art, as are suitable conditions for hybridization of e.g. Fluorophore 1 labeled probes with primers according to the invention.

Inventive primers are characterized in that they allow an enzymatic extension at its 3'-end, optionally, the inventive

Primers carry one or more modifications at their 3 'end which allow enzymatic extension of the 3' end complementary to the target. Such

Modifications are known to the person skilled in the art. Particularly preferred is such

Modification in a free hydroxy group of the terminal base at the 3 'end of the primer according to the invention, which is suitable for enzymatic extension.

Suitable means and routes for enzymatic extension of the primers according to the invention at their 3'-end complementary to the target are known to those skilled in the art. Such enzymatic extension can be mediated, for example, by use of a suitable DNA polymerase. Corresponding reaction conditions are known to the person skilled in the art. A Target Specific Sequence (TSS) is a nucleotide sequence that is complementary to a particular portion of the target sequence and allows for specific hybridization between the target strand and TSS-bearing primer or probe strand. Preferably, a TSS has a length of 3 to 100 nucleotides, more preferably from 3 to 50, most preferably from 5 to 35 nucleotides.

Another embodiment of the method is shown below:

Method for the real-time detection of nucleic acid targets as products of a nucleic acid amplification reaction by means of a primer / probe system comprising the steps:

a. Use of at least one primer which comprises at least one sequence specific to the nucleic acid target to be amplified (TSS) and which at the 3'-end allows enzymatic extension complementary to the target.

b. Introduction of a fluorophore 1 to the at least one primer from step a), preferably by means of covalent bonding to the at least one primer or by means of a probe labeled with fluorophore 1, which hybridizes specifically to the at least one primer.

c. Enzymatic extension of the at least one primer.

d. Use of at least one probe 2 which carries a fluorophore 2 at least at the 3 'end. In this case, the at least one probe 2 with the

Strand of the target sequence at the 3 'end extended at least one primer from step c), which carries the fluorophore 1, hybridize, so that Fluophore 1 and Fluophore 2 come in close proximity, that a fluorescence resonance energy transfer between the Fluophores 1 and 2 is possible.

e. Excitation of one of the two fluorophores by light of suitable wavelength, so that between the fluorophores 1 and 2 takes place a fluorescence resonance energy transfer.

f. Fluorescence optical detection of fluorescence. In a particular embodiment, the two different, each with a FRET donor or FRET acceptor labeled primer / probe oligonucleotides are constructed so that after hybridization by forming an intramolecular hairpin between a hairpin sequence (HBS) of the at least one primer and the HBS-complementary portion of the enzymatically extended at least one primer, the fluorophores 1 and 2 are in a proximity sufficient for the FRET to allow fluorescence of the acceptor during a PCR cycle. That is, one of the two fluorophores is excited by light of a suitable wavelength, so that a FRET takes place between fluorophore 1 and 2 which is detected by fluorescence optics. Such a sufficient proximity between the two fluorophores is present, for example, when the two fluorophores 1 and 2 are not more than 10 nucleotides apart, preferably not more than 5 nucleotides, very particularly preferably between 1 and 5 nucleotides. When the required proximity between fluorophore 1 and fluorophore 2 is mediated via a hairpin structure, the spatial proximity of the two fluorophores is independent of the actual nucleotide spacing on the linear sequence of the strand that hybridizes to probe 2. It does not matter whether fluorophore 2 comes to lie downstream or upstream of fluorophore 1 at the appropriate distance. Surprisingly, the FRET between fluorophore 1 and 2 is also not affected by the fact that according to the invention fluorophore 1 and fluorophore 2 are positioned on opposite strands.

A hairpin sequence (HBS) is a nucleotide sequence that has a sequence that is complementary to a downstream or upstream additional nucleotide sequence of the same strand molecule and that permits intramolecular hybridization of the two complementary strand regions. Thus an intramolecular so-called hairpin structure is formed. The HBS preferably has a sequence which is essentially identical to a sequence segment of the target, this target segment (also called complement to TBS 2) downstream of the target-specific binding site of the primer (the complement to TSS 1) in the direction of the enzymatic extension of the HBS molecule. carrying primer or probe is located. Preferably, a HBS has a length of 3 to 100 nucleotides, more preferably from 3 to 50, most preferably from 3 to 35 nucleotides, or from 3 to 20 nucleotides.

Such a method is described below: Method for the real-time detection of nucleic acid targets as products of a nucleic acid amplification reaction by means of a primer / probe system comprising the steps:

a) Use of at least one primer which comprises at least one target-specific sequence (TSS) for the nucleic acid to be amplified, as well as a hairpin formation sequence (HBS), which at the 3'-end permit enzymatic extension complementary to the target and the introduction of a fluorophore 1 through covalent bond or by binding a probe 1 allowed.

b) Introduction of at least one fluorophore 1 by covalent bonding to the at least one primer from step a) or by hybridization of a probe

1, which is labeled with fluorophore 1, with the at least one primer from step a).

c) Enzymatic extension of the primer at the 3'-end.

d) use of at least one probe 2 which carries a fluorophore 2 at least at the 3 'end, wherein the one probe hybridizes with the primer extended at the 3' end, so that fluophore 1 and fluophore 2 come into close proximity to one another Fluorescence resonance energy transfer between the fluorophores 1 and 2 is possible.

e) formation of at least one intramolecular or intermolecular hairpin by hybridization of the HBS of the at least one primer extended according to step c) with the section of the extended primer that is complementary to the HBS or with a section of the HBS - complementary section of

Probe 2.

f) excitation of one of the two fluorophores by light of a suitable wavelength, so that a fluorescence resonance energy transfer takes place between the fluorophores 1 and 2.

g) fluorescence optical detection of fluorescence. In a particular embodiment of the invention, the loop of the hairpin structure formed has a sequence length of at least 3 nucleotides, preferably more than 5 nucleotides, more preferably more than 10 nucleotides, more preferably more than 20 nucleotides, most preferably 25 or more Nucleotides, or wherein the sequence length of the loop structure is selected from a range of 25 to 60 nucleotides.

In the method according to the invention, one or more further probes may be used which may be labeled or unlabelled and which may specifically bind at least partially within the loop sequence of the hairpin structure formed to the strand of the extended at least one primer. Such additional probes may be hybridized with the extended at least one primer before, during or after use of the fluorophor 2 labeled probe 2. In this case, the sequence of the at least one further probe is selected so that when hybridization of this further probe to the extended primer, the hairpin structure is changed so that between FRF 1 and fluorophore 2 no more FRET can take place. This is e.g. then the case when the hairpin structure is destabilized by the binding of the at least one further probe or the Harrnadelstruktur is even dissolved. The destabilization of the hairpin or the dissolution of the hairpin can be achieved by overlapping the specific binding sequence of the at least one further probe at least partially with the stem region of the hairpin. The further probe may also overlap with the sequence of primer 1 extending into the hairpin.

In addition to the hairpin a preferably at the 3'-end blocked or at the 3'-end non-complementary probe (blocking probe), which preferably carries no fluorescent dye, hybridize and to dissolve the hairpin and cancel the required for a FRET distance between fluorophore 1 and 2 contribute. Alternatively or in combination with this probe, partial sequences of co-amplified target sequences, e.g. Hybridize pathogen sequences or internal standards in the hairpin.

By differential hybridization e.g. during melting curve recording, it is possible to identify sequence polymorphisms using at least one additional probe, preferably at least one unlabeled blocking probe or co-amplified target sequences, over wide sequence regions of the hairpin.

In a further embodiment of the invention, the at least one primer additionally has a tail which is unspecific to the target at the 5 'end. Fluorophore 1 can be used for Example at the specific or unspecific part of a first primer or on another primer. The arrangement of the fluorophores is exchangeable, so that fluorophore 1 can be present on a primer and fluorophore 2 on the probe or vice versa. Alternatively, fluorophore 1 and 2 can be incorporated into probes. In which direction FRET takes place or is detected has no influence on the method according to the invention, both directions are encompassed by the invention. Preferably, the fluorophores 1 and 2 are covalently bonded to primers and probes.

The invention is based on a novel primer / probe design, wherein in a very particularly preferred variant of the at least one primer, this has a partial sequence which is partially complementary to its 3 'region in the 5' region, so that it has an intramolecular Hairpin forms.

Preferably, the at least one primer may have the fluorophore 1 at the 5 'end, wherein the primer consists either only of a target-specific sequence or additionally has a nonspecific tail. After enzymatic extension and probe hybridization, the primer at the 5 'end partially or completely forms a hairpin with itself or to the primer sequence that is complementary to the target to detect the target, so that a FRET signal is produced.

In another embodiment of the invention, the 5 'end of the primer target is nonspecific and complementary to the 3' end of a probe which in turn hybridizes with its 5 'region to the target. This forms a hairpin-like loop structure between extended primer and probe. Fluorophore 1 at the 5 'end of the primer and fluorophore 2 preferably at the 3' end of the probe, which is thus blocked at the same time, come into close proximity through the loop formation, so that FRET takes place. Alternatively, the sequence-unspecific fluorophore-labeled 5 'end of the primer can also hybridize to the complementary, non-target specific end of the 5' end of the probe. Also in this case, FRET occurs when the probe carries fluorophore 2 in the region of the 5 'end. Of course, it is also possible that the primer is labeled in the region of the 5 'end with Fluophore 1 and the probe at the 5' end with Fluophore 2. Fluophore 2 can also be bound to the primer if the probe carries Fluophore 2. It is also contemplated that primer 1 is a probe primer consisting of a target sequence specific 3 'and a target sequence unspecific 5' portion and is specific for the PCR. On the other hand, a primer 2 which carries one of the two fluophores and is in sequence with the target sequence-unspecific section of the probe primer determines the sensitivity of the amplification. That's it possible, in a multiplexed approach using different probe primers and a universal primer 2 which inexpensively inserts a fluorophore into all amplicons. In this way, all other variants for multiplex use can be extended.

In a very preferred embodiment of the invention, the method is characterized in that it uses at least three primers. In this case, it comprises the following steps: In a first reaction, the use of a primer pair 1 and 2. The selected forward primer 1 comprises at least one target binding sequence 1 (TSS 1) and at least one hairpin sequence (HBS) and is designed so that TSS 1 can be attached to the can bind to target to be amplified. Subsequently, this primer 1 is extended complementary to the target, reverse primer 2 binds to the complementary target and is also extended. Primer 1 preferably contains a TSS 1 and a HBS. In one variant, it contains several HBS, which differ slightly to completely. Thus, in a reaction, multiple target sequences can be detected in total (e.g., detection of high risk and low risk human papillomaviruses). In a further reaction, a primer 3 is used which is identical or partially identical to the 5 'region of primer 1 but has no TSS. Primer 3 optionally carries fluorophore 1 at the 5 'end. It is designed so that it can form a hairpin 1 completely or partially with itself or with complementary portions of the primer 1 sequence. The conditions are adjusted so that with primer 2 and 3 an enzymatic extension and optionally an amplification takes place in one or more cycles. The at least one HBS of primer 1 binds to one or more further target binding portion (s), which now lies between extended primer 1 and the complementary sequence of primer 2, whereby a hairpin 2 is formed. If the amplificate is then brought into contact with the fluorophore 2 labeled probe, the FRET signal formed with the fluorophore 1 can be detected. In a preferred embodiment, the primer 3 carries fluorophore 1 and the probe with fluorophore 2 at the 3 'end hybridizes completely to the target sequence. Alternatively, the probe can hybridize with fluorophore 2 at the 3 'end via hairpin 2 and primer 1 at the target sequence. The loop sequence in hairpin 2 is preferably 10-100 nucleotides but can also be 50-1000 nucleotides depending on the analysis question.

Such a method according to the invention is shown below: a) in a first reaction,

Use of a primer pair 1 and 2, wherein the forward primer 1 at least one

Target binding sequence 1 (TSS 1) and at least one hairpin sequence (HBS), wherein the HBS is identical to a portion of the target sequence, which is located between the TSS 1 and the binding site of the reverse primer 2, the TSS 1 of the forward primer 1 to be amplified Target binds, then forward primer 1 is complemented enzymatically to the target, and then reverse primer 2 binds to the product of extension of forward primer 1 and is also enzymatically extended.

b) in a further reaction,

- Use of a primer 3, which

Is identical or partially identical to the 5 'region of primer 1,

• carries the fluorophore 1, and

Wherein primer 3 is constructed so that it can form a first intramolecular hairpin completely or partially with itself or with corresponding portions of the primer 1 sequence, and

Enzymatic extension of primer 3 using the extended reverse primer 2 from step a) as a template.

c) Use of at least one probe 2, which carries a fluorophore 2 at least at the 3 'end, wherein the probe 2 hybridizes with the extended primer 3 so that Fluophore 1 and Fluophore 2 come into spatial proximity such that a fluorescence resonance energy transfer between the fluors is possible.

d) evisceration of a second intramolecular hairpin in the extended primer 3 from step b) by hybridization of the at least one HBS of the original primer 1 sequence with the HBS complementary portion of the extended primer 3.

e) excitation of one of the two fluorophores by light suitable

Wavelength, so that between the fluorophores 1 and 2 takes place a fluorescence resonance energy transfer.

f) fluorescence optical detection of fluorescence.

In a particular embodiment of the invention, the loop of the hairpin structure 2 formed has a sequence length of at least 3 nucleotides, preferably more than 5 nucleotides, more preferably more than 10 nucleotides, more preferably more than 20 nucleotides, most preferably 25 or more nucleotides, or wherein the sequence length of the loop structure is selected from a range of 25 to 60 nucleotides.

In the method according to the invention, one or more further probes may be used which may be labeled or unlabeled and which may bind specifically to the strand of the extended primer 3 at least partially within the loop sequence of the hairpin structure 2 formed. Such additional probes may be hybridized with the extended primer 3 before, during, or after use of the fluorophor 2 labeled probe 2. In this case, the sequence of the at least one further probe is chosen so that when hybridization of this further probe to the extended primer 3, the Harrnadelstruktur is changed so that no more FRET can take place between fluorophore 1 and 2 fluorophore. This is e.g. then the case when the hairpin structure is destabilized by the binding of the at least one further probe or the Harrnadelstruktur is even dissolved. The destabilization of the hairpin or the dissolution of the hairpin can be achieved by overlapping the specific binding sequence of the at least one further probe at least partially with the stem region of the hairpin.

In addition, in hairpin 1 or 2, a preferably at the 3'-end blocked or at the 3'-end non-complementary probe (blocking probe), which preferably carries no fluorescent dye, hybridize and to dissolve the hairpin and cancel the required for a FRET distance between fluorophore 1 and 2 contribute. Alternatively or in combination with this probe, partial sequences of co-amplified target sequences such as pathogen sequences or internal standards can hybridize in hairpin 1 or 2. By differential hybridization, for example during the recording of melting curves, it is possible to identify sequence polymorphisms using at least one further probe, preferably at least one unlabeled blocking probe or co-amplified target sequences over wide sequence ranges of the hairpin.

In a further embodiment of the invention, the three primers and two probes can be used, each carrying FRET donor and acceptor (fluorophore 1 and 2), wherein the inserted probe 1 is primer-1 specific and the probe used is 2 target specific.

The fluorophores 1 and 2 used according to the invention are selected from

Fluorescent dye molecules, wherein the light which is absorbed and remitted by the first fluorophore is reabsorbed and remitted by the second second fluorophore spatially close to the first fluorophore after binding of the probe (s) to the first fluorophore. Preferably, various fluorophores serve as donor and acceptor, so that a FRET signal as

Occurrence of donor-stimulated fluorescence of the acceptor can be recorded.

Alternatively, the same dye can be used as donor and acceptor and the resulting depolarization of the fluorescence can be detected. The dyes may also be selected so that upon contact a non-radiative energy transfer takes place. Fluorophore 1 is preferably fluorescein and fluorophore 2 LC Red 640 or LC

Red 705 Examples of further preferred FRET pairs are:

Fluorophore A FRET partner B

Aminocoumarin fluorescein fluorescein rhodamine or tetramethylrhodamine

Fluorescein Cy3

Cy3 Cy5

Cy3 tetramethylrhodamine

Europium Cy5 terbium rhodamine

Bodipy Bodipy

Dansyl fluorescein

Naphtalene Dansyl

Ruthenium Cy5.

As already stated, according to the invention fluorophore 1 on a primer and

Fluorophore 2 are present on the probe or vice versa. The FRET pair also can

Probes bound. The FRET pairs used according to the invention achieve a high degree of multiplexing. Numerous fluorophores for the spectral range from 350 to 750 nm, that is from ultraviolet to infrared, are available for the process according to the invention.

The amplification reaction is preferably a homogeneous liquid-phase PCR, a multiplex PCR, an asymmetric PCR, a solid-phase PCR, an RT-PCR, an in situ PCR, an immuno-PCR or a nested PCR.

The method for real-time detection of nucleic acid targets as a product of a nucleic acid amplification reaction by means of the novel primer / probe systems for the detection of nucleic acids comprises (see Example 1): a) Use of at least one primer containing at least one target-specific sequence for the nucleic acid to be amplified (TSS) and a hairpin sequence (HBS) and which at the 3 'end allows an enzymatic extension complementary to the target and the

B) enzymatic extension of the primer at the 3'-end, c) formation of at least one intramolecular hairpin by hybridization of the HBS with the extended primer, whereby fluorophore

D) use of at least one probe 2 carrying a fluorophore 2 at least at the 3 'end, said at least one probe 2 hybridizing with the primer extended at the 3' end, e) excitation of one of the two fluorophores by light suitable

Wavelength so that a fluorescence resonance energy transfer takes place between the fluorophores 1 and 2 f) fluorescence optical detection of the fluorescence.

Preferably, different FRET pairs are used for the different amplicons or polymorphisms.

A preferred method by means of the primer / probe systems according to the invention comprises: a) Use of at least one primer which comprises at least one target specific sequence (TSS) and a hairpin formation sequence (HBS) for the nucleic acid to be amplified, and at the 3 'end allows enzymatic extension complementary to the target and mediates the introduction of a fluorophore 1 by covalent bonding or by binding a probe 1, b) enzymatic extension of the primer at the 3 'end, c) use of at least one probe 2, which is at least 3' -The End

Target sequence is nonspecific and carries a fluorophore 2, wherein the at least one probe 2 hybridizes with the primer extended at the 3 'end, d) formation of at least one intramolecular hairpin by hybridization of the HBS to the 3' end of the hybridized to the extended primer Probe 2, whereby fluorophore 1 is brought in close proximity to the fluorophore 2 of probe 2, e) excitation of one of the two fluorophores by light of suitable wavelength so that a fluorescence resonance energy transfer takes place between the fluorophores 1 and 2. f) fluorescence optical detection of the fluorescence.

A further preferred method by means of the primer / probe systems according to the invention comprises: a) use of at least one primer which comprises at least one target specific sequence (TSS) and a hairpin formation sequence (HBS) for the nucleic acid to be amplified, and an enzymatic at the 3 'end Extension allowed complementary to the target and mediates the introduction of a fluorophore 1 by covalent bonding or by binding a probe 1, b) enzymatic extension of the primer at the 3 'end, c) formation of at least one intramolecular hairpin by hybridization of the HBS with the extended primer d) binding of a blocking probe depending on the sequence of the hairpin e) use of at least one probe 2, which carries a fluorophore 2 at least at the 3 'end, wherein the at least a probe hybridizes to the primer extended at the 3'-end, f) prim One of the two fluorophores by light of appropriate wavelength, so that between the fluorophores 1 and 2 a

Fluorescence resonance energy transfer takes place g) Fluorescence optical detection of the fluorescence. If the melting temperature of the blocking probe of the hairpin sequence is lower than the melting temperature of the hairpin itself, the binding of the blocking probe leads to the opening of the hairpin already below the melting point of the hairpin. The hybridization properties and thus the properties of the hairpin sequence, in particular the occurrence of polymorphisms in the

Hairpin, can be detected by recording a melting curve.

A preferred primer / probe system comprises: a) three primers, wherein - a primer pair 1 and 2 function as forward and reverse primers and primer 1 comprises at least two sequences specific to the nucleic acid target to be detected (TSS 1 and HBS) and both allow enzymatic extension , and a primer 3 which is partially or completely identical to the 5 'region of primer 1, is also enzymatically extendible, and

Fluorophore 1, designed to be able to form a hairpin 1 wholly or partially with itself or with complementary portions of the primer 1 sequence, b) a probe bearing fluorophore 2 at the 3 'end and complete is specific for the target sequence, or specific for the target sequence and primer 1.

The invention may also be a primer / probe system comprising: a) three primers, wherein - a pair of primers 1 and 2 act as forward and reverse primers and wherein primer 1 comprises at least two sequences specific to the nucleic acid target to be detected (TSS) and both allow enzymatic extension, and

- Primer 3, which is partially or completely identical to the 5 'region of primer 1 and is also enzymatically extendable, b) two probes each carrying fluorophore 1 and 2 and which are completely specific for the target sequence, or for the Target sequence and primer 1 are specific.

The invention may also be a primer / probe system comprising: a) three primers, wherein

a primer pair 1 and 2 function as forward and reverse primers and primer 1 at least two for the Nucleic acid target specific sequences (TSS) to be detected and both allow enzymatic extension, and - primer 3 which is partially or completely identical to the 5 'region of primer 1 and which is also enzymatically extendable, b) two probes, each containing fluorophore 1 and 2 which are completely specific for the target sequence or specific for the target sequence and primer 1. c) a probe that binds wholly or partially in a hairpin 2 of one or more target sequences and has been modified at the 3 'end so that it can not be extended enzymatically.

The invention also provides a reagent kit for detecting nucleic acids comprising - at least one novel primer / probe system, suitable buffers and, if appropriate, further auxiliaries.

Buffers and auxiliaries are known to the person skilled in the art. They preferably comprise thermostable polymerase, dNTP mix, PCR buffer and optionally MgCl ₂ .

In the technical realization of nucleic acid assays for routine examinations are of particular importance, the homogeneous assay, the multiplex method and the so-called microarrays (also called gene or biochips). In a multiplex PCR according to the invention with the primer / probe system or the kit, a plurality of primer sets can be combined with the method described above, which allow the detection of several target sequences in one reaction batch on the basis of different target binding sequences (TSS) or HBS. Qualitative measurements are carried out by checking after a predefined number of amplification cycles whether the concentration of the nucleic acid molecules formed exceeds a certain threshold value. For quantification, this concentration is detected after each cycle and the number of cycles is determined until a certain threshold is reached. This number is a measure of the concentration of the desired nucleic acid in a sample.

With the aid of the system and method according to the invention, the target sequences in the probe binding region, in the primer binding region or in the stem of the hairpin (s) can preferably be distinguished by melting curve analysis for the simultaneous differentiation of a plurality of target sequences. In addition, for simultaneous discrimination of multiple target sequences, multiple primer / probe systems can be employed which differ at least partially or completely in the target sequence specific sections and emission wavelengths of the emitting fluorophores so that they can be differentially detected by multicolor fluorescence detection.

Furthermore, to detect multiple target sequences using allele-specific PCR, the target-specific primers can differ in the 3 'region and thereby be completely or incompletely complementary to the target sequence in the respective sample, as a result of which they are detected or not detected.

Furthermore, different detection systems can be used for the simultaneous detection of multiple target sequences, wherein the target-specific primers to be used can contain length polymorphisms and fluorescent labels.

In addition, the target sequences can be detected by real-time fluorescence analysis, by electrophoresis or by hybridization without amplification of the target sequence, by hybridization after amplification of the target sequence or by hybridization during amplification of the target sequence in solution or on a solid phase. Fluorescence-encoded bead arrays and spatially encoded biochips are used as preferred solid phases.

Real-time PCR owes its wide distribution to two properties that rarely fit together well in the lab routine: it is fast and at the same time precise. It takes less than two hours for a real-time cycler to duplicate and quantify the tiniest amounts of DNA. The whole thing works in high throughput. Up to 384 PCR reactions can simultaneously be performed by devices whose cycler blocks can be equipped with appropriate microtiter plates.

Real-time cycler are well known. They allow for detection of up to preferably five fluorescent dyes simultaneously for multiplexing applications, have heating blocks preferably in a four-pack for 96 or 384 well microtiter plates, show fast heating / cooling rates which allow eg 30 PCR cycles in 30 minutes, quantification software, melting point analysis and much more. The system and method of the present invention is also suitable for high throughput laboratories that have to process entire piles of samples, yielding up to 5,000 analyzes per day with corresponding state-of-the-art automated real-time PCR robots possible are. If no value is placed on real-time quantification and there is only interest in the end-point PCR, up to 30,000 samples can be processed in just under 3 hours using appropriate PCR robots.

With the system according to the invention, a prefabricated real-time PCR kit can be provided which contains all the necessary ingredients. Thus, the individual pipetting of buffer, nucleotide mix, reporter molecules, polymerases and primers is not necessary. For quantification in this (classical) method of real-time PCR, the melting point determination of the amplified DNA and the analysis of the threshold value of the fluorescence intensity (CT) are used.

According to the invention, the use of a primer / probe system according to the invention can be carried out in a device which comprises the probes immobilized as capture probes, for the simultaneous detection of target sequences during the amplification of the target sequences, wherein the randomly or systematically distributed in the reaction environment measuring points to which the target sequences the immobilized capture probes hybridize, imaging fluorescence optically detected, decoded and measured and the reaction environment is thermocyclically controlled, possibly by Peltier elements, air flow, water bath, heat radiation, induction or microwaves.

The performance of real-time detection can be accomplished using the probe systems of the invention but also using other probe systems, e.g. Molecular Beacon, FRET probes, TaqMan, Scorpions, GenePins or Amplifluor primers.

Advantages of the method according to the invention are:

A primer that can be used universally for different amplificates already carries a fluorophore. As a result, the fluorophores of the probes of different amplicons can be detected at low cost

(Example 3).

The dye-labeled probes and primers need not be polymorphic, since the polymorphisms can be detected by varying target binding sequences of unlabelled primer 1. This makes it possible to optimize the dye-labeled probes and primers for maximum detection sensitivity and to determine the specificity for the identification of sequence differences on the optimization of unlabeled primer limit (eg primer 1, Example 3), which is the cost of the

Optimization of PCR systems reduced.

However, polymorphic DNA cleavage can also be detected via inexpensive unlabelled probes which bind in hairpin 2 and cause a very efficient opening of hairpin 2, so that

Polymorphisms can be detected very specifically and thus reliably via melting curve courses.

Different amplicons can be obtained via inexpensive, unmarked

Probes are detected, which bind in hairpin 1 and cause a very efficient opening of hairpin 1, so that different

Amplikons very specific and thus can be detected safely.

By very large hairpins 2, the method is very variable because the probe binding sites can be placed on the target in optimal binding areas. - By very large hairpins 2, the method is very variable, since the use of multiple blocking probes large sequence areas can be examined for sequence changes, without the need for additional, labeled probes.

Through the use of multiple blockade probes, larger sequence regions within the hairpin can be easily examined for polymorphisms.

Since a fluorescent dye is covalently bound to the amplificate via a primer and thus only one probe must be used, higher sensitivities can be achieved, more stable readings can be obtained over a wider temperature range, making the system more robust than the classic two-probe system ,

The invention is shown schematically in FIGS. 1 to 5.

The invention will be explained in more detail with reference to drawings and exemplary embodiments, wherein it should not be limited thereto.

Fig. 1 - schematic representation of the use of the primer / probe system using a primer and a probe for the detection of a target polymorphism

Primer 1 carries a covalently attached 5'-end fluorophore 1 and a target binding sequence 1 and a hairpin sequence, wherein the Hairpin sequence for polymorphism detection. Primer 1 binds with the target binding sequence 1 of its 3 'end to the target sequence in a 1st position and is complementarily extended by a DNA polymerase under suitable conditions. After denaturation of the double strand, the 5 'end of primer 1 via the hairpin sequence (HBS) forms a hairpin with the target sequence 2 of the gene comprising the polymorphism. In addition, the probe, which carries fluorophore 2 at the 3 'end, binds to the complementary to the target-extended primer immediately following the hairpin formed. By irradiation of the reaction mixture, a fluorescence-mediated FRET is triggered with light of defined wavelength and an emission of fluorophore 2 is measured. This emission correlates with the amount of template in the reaction mixture which comprises the polymorphism. To better distinguish target sequences with polymorphism and target sequences without, which differ only slightly in their target sequence 2, a melting curve is created. If both target sequences are contained in the reaction mixture, two peaks result in the representation of the melting curve.

Fig. 2: Detection of a factor 2 polymorphism

A fragment of the factor 2 gene is amplified between primer 1 and primer 2, where primer 1 carries at its 5 'target sequence unspecific end a fluorophore 2 which is complementary to the 3' target sequence unspecific end of the probe. The 3 'end of the probe carries fluorophore 1. As soon as the probe binds to primer 1, which is complementary to the target, the target sequence unspecific ends of the primer and probe hybridize, with fluorophore 1 and 2 coming into close proximity to form FRET. A melting curve is generated to identify polymorphisms in the probe binding region. But it is equally possible to additionally use a probe 2, which hybridizes in the loop area and thereby prevents the formation of the loop and thus FRET (not shown). Polymorphisms in the binding region of probe 2 can be identified by recording the melting curve of probe 2.

Fig. 3: Schematic representation of a detection of a target sequence polymorphism

Part of a nucleotide sequence is amplified between primer 1 and primer 2. Primer 2 is target sequence-unspecific at the 5 'end, ie, a portion of the oligonucleotide does not bind to the target. This part contains a sequence section which is complementary to a sequence section farther in the 3 'direction on the target and can hybridize with it after amplification to form a hairpin. Further in the 5 'direction on primer 2 is a sequence section which provides a possibility of hybridization for a probe 2 into the Introduces amplificate. Probe 1 is complementary to the primer 2 which is complementary to the target. After hybridization to the hairpin, the fluorophores of probes 1 and 2 come into close proximity so that FRET can take place. Hybridization to the hairpin, however, is prevented in low temperature ranges because then probe 3 can bind to the hairpin structure and the hairpin is not closed. The Dehybridisierungstemperatur of probe 3 is dependent on the occurrence of polymorphism in the hairpin. By recording a melting curve, the polymorphism in the hairpin can be determined more closely. It is of course also possible for one of the two fluorophores to be bound to primer 1, in which case the primer-specific probe 2 and its binding region in primer 2 can be dispensed with (see FIG. 3b).

FIG. 4: Schematic representation of the characterization of nucleotide sequences by melting curve analysis with several blocking sense probes. Part of a nucleotide sequence is amplified between primer 1 and primer probe 2. The primer probe 2 is target-specific at the 5 'end, i.e., a portion of the oligonucleotide does not bind to the target. This part contains a sequence section which is complementary to a sequence section farther in the 3 'direction on the target and can hybridize with it after amplification to form a hairpin. Further in the 5 'direction on the primer probe is a sequence segment which introduces a hybridization opportunity for a probe 2 into the amplicon. Probe 1 is complementary to a portion of the target. After hybridization to the hairpin, the fluorophores of probes 1 and 2 come so close to each other that FRET can take place. At low temperatures, probes 3 through 6 may hybridize in the hairpin structure, preventing the hairpin from closing. When the temperature increases, the probes dehybridize 3 to 6 according to their specific (different) melting temperatures and there is the formation of a characteristic melting curve. If there is a polymorphism under a probe, the melting curve typically changes in this case.

5 is a schematic representation of the use of the primer / probe system using three primers and a probe for real-time detection and electrophoretic detection of polymorphisms. (A) Primer 1 comprises two target binding sequences 1 and 2 and a 5 'end, which corresponds to the sequence of primer 3. Primer 1 binds with the target binding sequence 1 of its 3 'end to the target sequence of the gene to be detected and is extended by a DNA polymerase under suitable conditions complementary to the target. After denaturation of the double strand primer 2 binds to the extended primer 1 and is complementarily extended.

(B) The resulting duplex is denatured so that (C) Primer 3 binds with fluorophore 1 at the 5 'end to the complementary end of Primer 1 and is extended. (D) The resulting double strand is denatured, so that during rehybridization a hairpin 1 is formed between target binding sequence 2 and target sequence 2. In addition, a hairpin 2 is formed between the 5 'end of the primer 3 sequence and primer 1. The probe, which bears a label with fluorophore 2 at the 3'-end, binds to the complementary to the target-extended primer 3 immediately after the hairpin 1.

(D1) For selective amplification of different target sequences, primer 1 differs in the target binding sequence 1 and by a length marker between primer binding sequence 1 at the 3 'end and primer sequence 3 at its 5' end of other primers 1 for other target sequences in the multiplex approach. By irradiation of the reaction mixture with light of defined wavelength, a fluorophore-mediated FRET is triggered and an emission of fluorophore 2 is measured at a defined wavelength. This emission correlates with the amount of template in the reaction mixture which comprises the polymorphism. To better distinguish target sequences with polymorphism and target sequences without, which differ only slightly in their target sequence 2, a melting curve is created. If both target sequences are contained in the reaction mixture, two peaks result in the representation of the first derivative of the melting curve. Due to different length markers in different primers 1 (see D1?), The amplificates can additionally be detected or distinguished by gel electrophoresis or mass spectroscopy.

example 1

Detection of the pathogen herpes simplex - \ / ^'\ rus in a sample a volume of 5 ul from swabs of cold sores purified sample DNA becomes

15 μl of the master mix of the following composition:

Primer 1 400 nM

Primer 2 30O nM

Probe 100 nM dissolved in Taq DNA polymerase, nucleotides, magnesium chloride, Tris / HCl buffer and Tween 20. The reaction batch is pipetted into a LightCycler capillary and then transferred to the LightCycler and thermocyclically amplified. The method is shown schematically in FIG. Primer 1 carries a covalently bound fluorescein at the 4 th nucleotide counted from the 3 'end, binds with its target binding sequence 1 at the 3' end to the target sequence of the herpes s / mp / ex-virus genome and is by a DNA polymerase extended under suitable conditions complementary to the target. After denaturation of the double strand, the probe bearing a rhodamine label at the 3 'end can hybridize to the primer extended to complement the target. By irradiation of the reaction mixture with light having a wavelength of 480 nm, a fluorescein-mediated FRET is triggered and the emission at 560 nm correlates with the amount of template present in the reaction mixture.

After completion of the amplification, a melting curve is recorded for all samples which have a sufficiently high measurement signal.

sequences

Primer 1: 5'-TOAAGGG CCA GGC GCT TGT TGG TGT A-3 '(SEQ ID NO: 1) G at position 2 (highlighted in gray) covalently carries a FITC molecule.

Primer 2: 5'-CAT CAC CGA CCC GGA GAG GGA C-3 '(SEQ ID No: 2)

Probe: δ'-GCAACTCTCTCAGGGCCAGGCGG-S'-rhodamine (SEQ ID No: 3)

Example 2: Detection of a factor 2 polymorphism

A volume of 5 μl of sample blood purified from patient's blood or reamplifcation-prediluted amplicon is added to 15 μl of master mix of the following composition: Primer 1 (FITC-modified) 300 nM Primer 2 30O nM

Probe 10O nM

dissolved in Taq DNA polymerase, nucleotides, magnesium chloride, Tris / HCl buffer and Tween 20. The reaction batch is pipetted into a LightCycler capillary and then transferred to the LightCycler and thermocyclically amplified. The method is shown schematically in FIG. Primer 1 carries a Cy5 covalently attached at the 5 'end and a target binding sequence in the 3' region and a probe binding sequence in the 5 'region. With the target binding sequence, primer 1 binds to the target and is extended by a DNA polymerase under appropriate conditions complementary to the target, the factor 2 gene. After denaturation of the double strand, the 5 'end of primer 1 forms a hairpin-like loop with the target unspecific 3' region of the probe, the 3 'end of which was blocked by fluorescein labeling against polymerase extension. By irradiation of the reaction mixture with light having a wavelength of 480 nm, a fluorescein-mediated FRET is triggered and an emission of Cy5 at 640 nm is measured. This emission correlates with the amount of template in the reaction mixture which comprises the polymorphism. To better distinguish target sequences with polymorphism and target sequences without, which differ only slightly in their target sequence 2, a melting curve is created. If both target sequences are contained in the reaction mixture, two peaks result in the representation of the melting curve. The sequence polymorphism is preferably detected in the target-specific region of the probe. If there is a mutation of the target sequence in the probe binding region, there is a mismatch in the probe binding, so that the melting temperature of the probe changes. After completion of the amplification, a melting curve is recorded for all samples which have a sufficiently high measurement signal in order to find changes in the melting behavior of the probe and thus of any mutations.

sequences

Primer 1: Cy5-5'-GTCGTCGGCCTGGAACCAATCCCGTGAAAG-3 '

(SEQ ID No: 4)

Primer 2: 5'-CCA GGT GGT GGA TTC TTA AGT C-3 '(SEQ ID No: 5) Probe: 5'-CATTGAGGCTCGCTGAGAGTGCCGACGAC-3'-FITC (SEQ ID No: 6)

Example 3:

Detection of Target Sequence Polymorphism A volume of 5 μl of purified sample DNA becomes the following 15 μl of master mix

Composition given:

Primer 1 300 nM

Primer 2 30O nM

Probe 1 (FITC modified) 400 nM Probe 2 (LC Red 640 modified) 400 nM

Probe 3 500 nM dissolved in Taq DNA polymerase, nucleotides, magnesium chloride, Tris / HCl buffer and Tween 20. The reaction batch is pipetted into a LightCycler capillary and then transferred to the LightCycler and thermocyclically amplified. The method is shown schematically in FIG. Both primers are not labeled with fluorescent dyes. Primer 2 serves on the one hand as a primer (sequence region at the 3'-end) and on the other hand to introduce the complementary sequence for hairpin stem formation and for binding the probe 2 (FRET acceptor) in the amplicon. The amplicon produced by PCR forms a 20-base hairpin structure on which probe 3 can hybridize. In this case, probe 3 covers a mutation (x) to be detected. In addition, a labeled with a fluorescent dye probe 1 (FRET donor, eg fluorescein) in close proximity (preferably 0 to 10 bases distance to the first base of the hairpin strain) bind to the stem of the hairpin as well as probe 2 (FRET acceptor, eg LC Red -640) whose fluorescence label is at the opposite end of probe 1 (eg probe 1 5'-labeled, probe 2 3'-labeled). When the hairpin is closed, the probes labeled with fluorescent dyes come in close proximity to one another and a fluorescence signal of the FRET acceptor can be detected upon excitation with a suitable wavelength.

By also present in the reaction mixture probe 3, an opening of the hairpin is forced at low temperatures, since probe 3 can hybridize in the hairpin structure, that the closing of the hairpin is prevented. Only after increasing the temperature (e.g., when taking a melting curve) does probe 3 hybridize and the hairpin closes (increase in fluorescence signal). In the presence of a base exchange in the hairpin area (mutation to be detected), the probe dehybridizes correspondingly earlier and leads to a changed signal course. The changes can easily be detected with a melting curve analysis (double peak in the presence of wild type and mutation template, heterozygous constellation).

sequences

Primer 1: B'-CTGAATTCTTAGGTGGTGGACC-S ¹ (SEQ ID NO: 7) primer 2 5 '-GATCCAAGTCCAAACGTCAACCCATCTGACTACGCTGAGTGTTCCCAATAAAAGTGACTC-B'

(SEQ ID NO: 8)

Probe 1 (fluorescein-modified): Fiuorescem-5 ^■ -TAGTCAGATGGGTTGACGTTTGGACTTGGATC-3 ^■

(SEQ ID NO: 9)

Probe 2 (LC-Red-640-modified): 5 ^■ -CCATGAATAGCACTGGGAGCATTGAGGCT-B ^■ -Lc-Red640 (SEQ ID No: 10)

Probe 3: S '-GTCACTTTTATTGGGAAC-B' (SEQ ID NO: ii) Example 4:

Characterization of nucleotide sequences by melting curve analysis

A volume of 5 μl of purified sample DNA for amplification intended for reamplification is added to 15 μl of master mix of the following composition: Primer 1 300 nM

Primer 2 30OnM

Probe 1 (fluorescein-modified) 400 nM probe 2 (LC-Red-640-modified) 400 nM probe 3 to 6 50 oM in Taq DNA polymerase, nucleotides, magnesium chloride, Tris / HCl buffer and Tween 20 were dissolved.

The reaction batch is pipetted into a LightCycler capillary and then transferred to the LightCycler and thermocyclically amplified. The method is shown schematically in FIG. Both primers are not labeled with fluorescent dyes. Primer probe 2 serves on the one hand as a primer (sequence region at the 3 'end) and on the other hand to introduce the complementary sequence for hairpin stem formation and for binding the probe 2 (FRET acceptor) into the amplificate. The amplicon produced by PCR forms a hairpin structure of 66 bases length at which probes 3 to 6 can hybridize. Upon complete hybridization of probes 3 to 6, hybridization of the hairpin stem is prevented, and thus FRET can not occur upon excitation with 480 nm wavelength light. Upon a temperature increase in the course of recording a melting curve, the probes dehybridize in a defined order and depending on the sequence of the amplicon formed. If sequence changes in the probe binding region compared to the wild-type sequence are present, a modified melting curve is obtained, which allows conclusions to be drawn about the sequence region which has changed.

sequences

Primer 1: S'-CTGAATTCTTAGGTGGTGGACC-S ¹ (SEQ ID NO: 12) Primer Add 2: 5'-GATCCAAGTCCAAACGTCAACCCATCTGACTACTGGCGCTGAGTTTGTCGTTGTTCCGTC-B '(SEQ ID No: 13)

Probe 1 (fluorescein-modified): 5 ^■ -fluorescence-τAGτcAGATGGGττGACGτττGGAcττGGAτc-3 ^■

(SEQ ID No: 14)

Probe 2 (LC-Red-640 modified): s ^■ -ccATGAATAGCAcτGGGAGCAττGAGGcτ-Lc-Red640-3 ^■ (SEQ ID No: 15)

Probe 3: 5'-GACGGAACAACGACAA-3 '(SEQ ID NO: i6)

Probe 4: 5'-GGTCACTTGGATTGG-3 '(SEQ ID NO: 17)

Probe 5: 5'-CAGCGGCAGCGACAA-3 '(SEQ ID NO: 18) Probe 6: S '-GTCACTTTTATTGGGAAC-B' (SEQ ID NO: 19)

Example 5:

Simultaneous detection of herpes simplex DNA and internal standard in a sample A volume of 5 μl of sample blood purified from patient blood is added to 15 μl of master mix of the following composition:

Primer 1 a 10 nM

Primer 1 b 1 O nM

Primer 2 30O nM Primer 3 (FITC-modified) 30O nM

Probe 1 10O nM

Probe 2 10O nM

Internal standard 100 fM dissolved in Taq DNA polymerase, nucleotides, magnesium chloride, Tris / HCl buffer and Tween 20.

The reaction mixture is pipetted into a LightCycler capillary and then into the

LightCycler transferred and thermocyclic amplified. The procedure is schematic in

Fig. 3 shown. Upon completion of the amplification, a melting curve is recorded for all samples having a sufficiently high measurement signal to detect amplicon of the internal standard and, for HSV positive samples, HSV amplicon.

sequences

Primer 1a: 5'-actgcacgctcacacggacg gtgcagtaagtcct CAT CAC CGA CCC GGA GAG GGA C-3 '(SEQ ID No: 20)

Primer 1 b: 5'-actgcacgctcacacggacgttgtgtgtgtgtgtgtgtgt gttgttgttg ttgttgttgt gcgtgcagt gcggtccac CATCACCGAC CCGGACAGCC CA-3 '(SEQ ID No: 21) primer 2: δ'-GGG CCA GGC GCT TGT TGG TGT A-S' (SEQ ID No: 22)

Primer 3: FITC-5'-actgcacgctcacacggacg-3 '(SEQ ID No: 23)

Probe 1: 5'-tcagttcggcggtgaggaca-3'-rhodamine (SEQ ID No: 24)

Internal standard: 5'-CATCACCGAC CCGGACAGCC CACCAGCGTG GACCGCTGT CCT CAC CGC CGA ACT GA GGCCAG GCGGTGAAGG GCAATCAGCT GTTGCCCGTC TCGCTGGTGA AAAGAAAAAC CACCCTGGCG

CCCAATACGC AAACCGCCTA CACCAACAAG CGCCTGGCCC-3 '(SEQ ID NO: 25) Legend to the pictures

Primer = HBS

Complementary to the primer target sequence 2

Probe Hybridized Nucleic Acid Double Strand

Fluorophore 1

stimulation

emission

X o _d er - = & ► polymorphism in the target SA FRET probe 2

Claims

claims

A method for the real-time detection of nucleic acid targets as products of a nucleic acid amplification reaction by means of a primer / probe system, characterized in that it comprises the following steps:

a) Use of at least one primer which comprises at least one target-specific sequence (TSS) for the nucleic acid to be amplified and a hairpin-forming sequence (HBS) which allows enzymatic extension complementary to the target at the 3 'end and the introduction of a fluorophore 1 through covalent bond or by binding a probe 1 allowed.

b) introduction of at least one fluorophore 1 by covalent bonding to the at least one primer from step a) or by hybridization of a probe 1 which is labeled with fluorophore 1, with the at least one primer from step a).

c) Enzymatic extension of the primer at the 3'-end.

e) formation of at least one intramolecular or intermolecular hairpin by hybridization of the HBS of the at least one primer extended according to step c) with the section of the extended primer complementary to the HBS or with a section of probe 2 complementary to the HBS. f) excitation of one of the two fluorophores by light of a suitable wavelength, so that a fluorescence resonance energy transfer takes place between the fluorophores 1 and 2.

g) fluorescence optical detection of fluorescence.

2. The method according to claim 1, characterized in that the at least one primer additionally has a nonspecific nucleotide sequence to the target at the 5 'end.

3. The method according to any one of claims 1 to 2, characterized in that probe 2 and optionally probe 1 to the strand formed by extension according to claim 1, step c) containing the at least one primer.

4. The method according to any one of claims 1 to 3, characterized in that the fluorophore 1 is covalently bonded to the at least one primer or to the probe 1.

5. The method according to any one of claims 1 to 4, characterized in that the

Fluorophore 2 is covalently bound to probe 2.

6. The method according to any one of claims 1 to 5, characterized in that the at least one primer comprises a sequence specific to the target sequence to be amplified (TSS) and in the 5 'region to its 3' region partially complementary HBS, so that the primer can form a hairpin in itself.

7. The method according to any one of claims 1 to 6, characterized in that the fluorophore 1 is not bound to the TSS or the HBS of the at least one primer.

8. The method according to any one of claims 1 to 7, characterized in that the at least one primer has the fluorophore 1 at its 5 'end.

9. A method for real-time detection of nucleic acid targets as products of a nucleic acid amplification reaction using a primer / probe system comprising the steps: a) in a first reaction,

Use of a primer pair 1 and 2, wherein the forward primer 1 at least one target binding sequence 1 (TSS 1) and at least one

Hairpin forming sequence (HBS), wherein the HBS is identical to a portion of the target sequence extending between the TSS 1 and the binding site of the

Reverse primer 2 is located, - the TSS 1 of the forward primer 1 to the to be amplified

Target binds, then forward primer 1 is complemented enzymatically to the target, and then reverse primer 2 binds to the product of extension of forward primer 1 and is also enzymatically extended.

b) in a further reaction,

Use of a primer 3 which is identical or partially identical to the 5 'region of primer 1,

• carries the fluorophore 1, and

• wherein primer 3 is designed to be a first intramolecular hairpin completely or partially with itself or with appropriate portions of the hairpin

Primer can form 1-sequence, and

c) use of at least one probe 2 which is at least on the 3 '

End carries a fluorophore 2, wherein the probe 2 hybridized with the extended primer 3, so that Fluophore 1 and Fluophore 2 get in close proximity, that a fluorescence resonance energy transfer between the fluorophores is possible.

e) excitation of one of the two fluorophores by light suitable

f) fluorescence optical detection of fluorescence.

10. The method according to claim 9, characterized in that primer 3 carries the fluorophore 1 at the 5 'end.

1 1. A method according to any one of claims 9 and 10, characterized in that primer 3 carries the fluorophore 1 and the probe 2 via the second intramolecular hairpin from step c) of claim 9 exciting time, simultaneously to the original forward primer 1 sequence and to a downstream of the 3 'end of the second hairpin portion of the extended primer 3, which is complementary to a portion of the target sequence, which between

TSS 1 and the binding site of the reverse primer 2 is located.

12. The method according to any one of claims 1 to 1 1, characterized in that probes 1 and 2 are used, wherein probe 1 carries the fluorophore 1 and probe 2 is the fluorophore 2 and probe 1 primer 1 -specific and probe 2 target specific.

13. The method according to any one of claims 1 to 12, characterized in that the fluorophores 1 and 2 are selected from fluorescent dye molecules, wherein the light, which is absorbed by the first fluorophore and remitted, from the second, after

Binding of the probe 2 to the first fluorophore spatially close fluorophore 2 is re-absorbed and remittiert or is transmitted without radiation via contact of the dye molecules.

14. The method according to any one of claims 1 to 13, characterized in that the following fluorophore pairs are used, wherein either fluorophore 1 can be selected from the group of the fluorophore A and fluorophore 2 then represents the corresponding FRET partner B, or vice versa: Fluorophore A FRET partner B

Fluorescein LC Red 640 or LC Red 705

Aminocoumarin fluorescein

Fluorescein rhodamine or tetramethylrhodamine fluorescein Cy3

Cy3 Cy5

Cy3 tetramethylrhodamine

Europium Cy5

Terbium Rhodamine Bodipy Bodipy

Dansyl fluorescein

Naphtalene Dansyl

Ruthenium Cy5.

15. The method according to any one of claims 1 to 14, characterized in that the enzymatic extension reaction is a homogeneous liquid-phase PCR, a multiplex PCR, an asymmetric PCR, a solid-phase PCR, an RT-PCR, an in situ PCR, a immuno-PCR or a nested PCR.

16. The method according to any one of claims 1 to 15, characterized in that for the simultaneous differentiation of multiple target sequences, the target sequences in the probe binding region, in the primer binding region, in the trunk of the hairpin (s) are distinguished by melting curve analysis or by the use of multiple fluorophores or length markers.

17. The method according to claim 1 to 16, characterized in that bind to the sequence or parts of the sequence of hairpin 2, one or more probes, said binding to cancel the hairpin and necessary for FRET and radiationless energy transfer small distance between fluorophore 1 and Fluorophore 2 leads.

18. A primer / probe system for the detection of nucleic acids comprising:

a) a primer comprising at least one sequence specific for the nucleic acid target to be detected and allowing an enzymatic extension at its 3 'end, this primer being labeled with the fluorophore 1, b) a probe which carries a fluorophore 2 at the 3 'end and which binds to the enzymatically extended primer from a).

19. primer / probe system according to claim 18 comprising: a) three primers, wherein

a primer pair 1 and 2 function as forward and reverse primers and wherein primer 1 comprises at least two sequences specific for the nucleic acid target to be detected and both primers 1 and 2 allow enzymatic extension, and

Primer 3, which is partially or completely identical to the 5 'region of primer 1, is also enzymatically extendible and carries fluorophore 1, being designed to form a hairpin 1 wholly or partially with itself, with complementary segments b) a probe which carries fluorophore 2 at the 3'-end and which is completely specific for the target sequence, or which is specific for the target sequence and primer 1. The primer can form the primer 1 or complementary sections of the target.

20. primer / probe system according to claim 18 comprising: a) three primers, wherein

Primer 3, which is partially or completely identical to the 5 'region of primer 1, is also enzymatically extendable, b) two probes, each carrying fluorophore 1 and 2, which are completely specific for the target sequence, or which are specific for the target sequence Target sequence and primer 1 are specific.

21. reagent kit for the detection of nucleic acids comprising a) at least one primer / probe system according to any one of claims 18 to 20, b) suitable buffers and optionally further excipients.

22. Use of a primer / probe system according to any one of claims 18 to 20 or a reagent kit according to claim 21 for the simultaneous differentiation of multiple target sequences, wherein a plurality of primer / probe systems are used, at least partially or completely in the target sequence specific sections and the emission wavelengths distinguish the emitting fluorophores so that they can be differentially detected by multicolor fluorescence detection.

23. Use of a primer / probe system according to any one of claims 18 to 20 or a reagent kit according to claim 21 for the detection of several

Target sequences, wherein the target-specific primers differ in the 3 'region and thereby are completely or incompletely complementary to the target sequence in the respective sample and thereby detected or not detected.

24. Use of a primer / probe system according to any one of claims 18 to 20 or a reagent kit according to claim 21 for the simultaneous detection of multiple target sequences using different detection systems, wherein the target-specific primer to be used contain length polymorphisms and may contain fluorescent labels.

25. Use of a primer / probe system according to any one of claims 18 to 20 or a reagent kit according to claim 21, wherein the target sequences by real-time fluorescence analysis, by electrophoresis or by hybridization without amplification of the target sequence, by hybridization after amplification of the target sequence or by hybridization during the Amplification of the target sequence can be detected.

26. Use of a primer / probe system according to any one of claims 18 to 20 or a reagent kit according to claim 21, in a microarray which the

Probes as capture probes or a subset of a primer immobilized as capture primer comprises.